Multi-protein complexes, which are the product of protein—protein interactions, participate in a variety of cellular processes. Such exemplary cellular processes include, for example, cell signaling, gene regulation, protein assembly and degradation, and mechanical events such as sarcomere shortening. Conserved structural motifs in many proteins have evolved to facilitate the interaction of specific proteins in the assembly of multi-protein complexes. The tetratricopeptide repeat (TPR) domain is one such structural motif that was originally identified by sequence comparisons among yeast proteins (Hirano et al., Cell, 60:319–328 (1990), Sikorski et al., Cell, 60:307–317 (1990)). The TPR domain contains a 34-amino acid structural motif with a loose consensus that is present, usually as multiple tandem repeats, in proteins with many cellular functions, including mitosis, transcription, protein transport, and development (Lamb et al., Trends in Biochemical Sciences, 20:257–259 (1995)). Structural analysis of the TPR domain demonstrates that it forms two α-helical regions separated by a turn, such that opposed bulky and small side chains form a “knob and hole” structure (Hirano et al., Cell, 60:319–328 (1990)). It is thought that a hydrophobic surface of this particular TPR domain mediates protein—protein interactions between TPR- and non-TPR-containing proteins.
TPR-containing proteins are typically known to play a diverse and important role in cellular function. Several TPR-containing proteins are known to participate in interactions with the major members of the heat shock protein family, Hsp70, Hsc70, and Hsp90. It is believed that these TPR-containing proteins are necessary for appropriate regulation of protein folding and transport. The TPR domains of protein phosphatase 5, cyclophilin 40 (CyP-40), and FKBP52 are known to mediate binding of these proteins to Hsp90 and assist in trafficking of nuclear hormone receptors (J. E. Kay, Biochem. J., 314:361–385 (1996)). A different group of TPR-containing proteins are known to interact with Hsc70 and Hsp70. Hsc70-Interacting Protein (HIP), also known as p48, binds to the ATPase domain of Hsc70, stabilizes the ADP-bound conformation, and increases the affinity for substrate proteins (Höhfeld et al., Cell 83:589–598 (1995)). Hsc70–Hsp90-Organizing Protein (HOP), also known as p60 or Sti1, serves as a coupling factor that facilitates the cooperation between Hsc70 and Hsp90, although it does not directly assist in chaperoning functions (Schumacher et al., J. Biol. Chem., 269:9493–9499 (1994)). In contrast to HIP, HOP interacts with the carboxy-terminal domain of Hsc70 (Demand et al., Mol. Cell. Biol., 18:2023–2028 (1998)).
In addition to fHP, at least two other proteins are known to regulate the reaction cycle of mammalian Hsc70 and Hsp70. Hsp40 stimulates the ATPase activity of Hsc70 and thus promotes the conversion of ATP-bound, low substrate-affinity Hsc70 to ADP-bound, high substrate-affinity Hsc70 (J. Höhfeld, Biol. Chem., 379:269–274 (1998)). The reverse reaction cycle, which involves exchange of ATP for ADP and loss of substrate affinity, was recently shown to be facilitated by the anti-apoptotic protein BAG-1 (Zeiner et al., EMBO J., 16:5483–5490 (1997)). Whereas HIP inhibits this reverse reaction cycle and stabilizes the ADP-bound conformation, no cellular inhibitors of the forward reaction cycle of the binding of a heat shock protein to a substrate to form a protein—protein complex have yet been identified.
Thus, there is a need for further understanding cellular components that regulate and interact with heat shock proteins, and identifying such cellular components.